Monday, 8 December 2014

New research paves the way for nano-movies of biomolecules

An international team, including scientists from Arizona State
University, the University of Wisconsin-Milwaukee (UWM), and Germany’s
Deutsches Elektronen-Synchrotron (DESY), have caught a light sensitive
biomolecule at work using an X-ray laser. Their new study proves that
high speed X-ray lasers can capture the fast dynamics of biomolecules in
ultra slow-motion, revealing subtle processes with unprecedented
clarity.

Ray of Light:A new generation of ultrafast
X-ray lasers is redefining the field of X-ray crystallography, revealing
never-before-seen features and dynamic processes.

"This work paves the way for movies from the nano-world with atomic
resolution," said Professor Marius Schmidt from UMW, corresponding
author of the new paper, which appears in the Dec. 4 issue of the
journal Science.

Study co-author Petra Fromme, professor in ASU’s
Department of Chemistry and Biochemistry, echoes the importance of the
new study: “This paper is very exciting as it is the first report of
time resolved studies with serial femtosecond crystallography that
unravels details at atomic resolution,” said Fromme. “This is a huge
breakthrough toward the ultimate goal of producing molecular movies that
reveal the dynamics of biomolecules with unparalleled speed and
precision.”

A femtosecond is a quadrillionth of a second, an
almost unfathomably brief duration. Around 100 femtoseconds are required
for a ray of light to traverse the width of a human hair.

The technique of X-ray crystallography allows
researchers to probe atomic and molecular structure, by exposing
crystals to incident X-rays that diffract from the sample in various
directions. Careful measurement of X-ray diffraction angles and
intensities allows a three-dimensional portrait of electron densities to
be constructed—information used to define atomic structure.

The technique has been an invaluable tool for
investigating the structure and function of a broad range of
biologically important molecules, including drugs, vitamins, proteins
and nucleic acids like DNA.

But just as shutter speed determines a camera’s
ability to capture action of very short duration, so X-ray lasers must
deliver extremely brief pulses of light to capture fine structure and
dynamic processes at the atomic level. Some of the phenomena researchers
wish to explore take place in mere quadrillionths of a second. A new
generation of ultrafast lasers like the Linac Coherent Light Source
(LCLS) at SLAC National Accelerator Laboratory (used in the current
study) are redefining the field of X-ray crystallography.

The researchers used the photoactive yellow protein
(PYP) as a model system. PYP is a receptor for blue light that is part
of the photosynthetic machinery in certain bacteria. When it catches a
blue photon, it cycles through various intermediate structures as it
harvests the energy of the photon, before returning to its initial
state. Most steps of this PYP photocycle have been well studied, making
it an excellent candidate for validating a new method.

For their ultrafast snapshots of PYP dynamics, the
scientists first produced tiny crystals of PYP molecules, most measuring
less than 0.01 millimeters across. The dynamics of these microcrystals
were captured in exquisite detail when the world’s most powerful X-ray
laser at SLAC was trained on them. Initiation of their photocycle was
triggered with a precisely synchronised blue laser pulse.

Thanks to the incredibly short and intense X-ray flashes of the LCLS,
the researchers could observe different steps in the PYP photocycle
with a resolution of 0.16 nanometers, by taking snapshots of X-ray
diffraction patterns. The spectacular time resolution afforded by the
technique allows researchers to detect changes in the atomic-scale
conformation of PYP molecules as they switch back and forth between
light and dark states.

Part of the ASU team of scientists involved in the work: (from left
to right) Christopher Kupitz, Dingjee Wang, Nadia Zatsepin, John Spence,
Petra Fromme and Raimund Fromme. Other members of the ASU team (not in
the photo) include Bruce Doak, Uwe Weierstall, Ingo Grotjohann,
Tzu-Chiao Chao, Mark Hunter and Richard Kirian.

The investigation not only reproduced what was
already known about the PYP photocycle, thereby validating the new
method, it also imaged delicate phenomena in much finer detail. Thanks
to the high temporal resolution, the X-ray laser could in principle
study steps in the cycle that are shorter than 1 picosecond (a
trillionth of a second) - too fast to be captured with previous
techniques. The ultrafast snapshots can be assembled into a movie,
detailing the dynamics in ultra slow-motion.

“This is far more than a proof of concept for time
resolved crystallography. LCLS can use micron size crystals and
therefore have an unmatched light initiation efficiency to explore
uncharted territory in the dimension of time resolution of molecular
reactions,” Raimund Fromme stated, an ASU associate research professor
participating in this project.

"This is a real breakthrough," emphasizes co-author
professor Henry Chapman from DESY. "Our study is opening the door for
time resolved studies of dynamic processes, providing an unprecedented
window on subtle transformations at the atomic scale."

John Spence, director of science for the STC at ASU,
stresses the importance of studying delicate life processes by means of
new tools capable of extreme spatial and temporal resolution:

"When combined with previous work, it is remarkable
now to be able to assemble a true molecular movie of the photocycle of
this blue light detector in bacteria at atomic resolution, with the
intermediate structures appearing and fading in the correct sequence. It
is a huge step forward, which will also aid research on artificial
photosynthesis,” he says. “It builds on our earlier work at LCLS, and is
supported by our NSF Science and Technology Center for the use of X-ray
lasers in biology."

The new research advances the ASU team’s pre-existing
investigations, highlighting the first time-resolved serial
femstosecond crystallography studies on Photosystem II. The study on
PYP now shows that this method can unravel previously undetected details
at the atomic level.

The ASU team involved in this study includes four
faculty and their research teams (John Spence, Uwe Weierstall, Petra
Fromme and Raimund Fromme) from the Departments of Physics and Chemistry
and Biochemistry who are members of the new Center for Applied
Structural Discovery at the Biodesign Institute. The ASU team
contributed to many aspects of the study, which range from experimental
planning to the application of injector technology, growth and
biophysical characterization of the PYP microcrystals and data
evalution.

The ASU team also includes the graduate students
Christopher Kupitz,Chelsie Conrad, Jesse Coe, Shatabdi Roy-Chowdhury,
who worked on the growth and biophysical characterization of the PYP
crystals at ASU and on site at LCLS, the graduate students Daniel James
and Dingjie Wang, who worked on sample delivery as well as the research
scientist Nadia Zatsepin and the graduate student Shibom Basu, who
worked on “on the fly” data evaluation.

“Since the sample injector developed at ASU allows
for continuous sample replenishment, the X-ray laser always probes
fresh, undamaged crystals, allowing us to make molecular movies of
irreversible reactions,” says Research Professor Uwe Weierstall.
Further, X-ray lasers typically investigate very small crystals that
often are much easier to fabricate than larger crystals. In fact, some
biomolecules are so hard to crystallise that they can only be
investigated with an X-ray laser.

“This is the highest resolution X-ray laser dataset
we’ve worked with – these tiny crystals were of very high quality,” adds
research scientist Nadia Zatsepin. “It was very satisfying to see such
high resolution electron densities by the second day of our experiment,
but to then also see such strong signals from the changes in the
structure was even more exciting,”

The small crystal size is also an advantage when it
comes to kick-starting molecular dynamics uniformly across the sample.
In larger samples, the initiating optical laser pulse is often quickly
absorbed in the sample, which excites only a thin layer and leaves the
bulk of the crystal unaffected.

The PYP microcrystals were perfectly matched to the
optical absorption so that the entire crystal was undergoing dynamics,
which in turn allows sensitive measurements of the molecular changes by
snapshot X-ray diffraction.

Taken together, X-ray laser investigations can offer
previously inaccessible insights into the dynamics of the molecular
world, complementing other methods. Using the ultra slow-motion, the
scientists next plan to elucidate the fast steps of the PYP photocycle
that are too short to be seen with previous methods.

In the future, ultrafast laser crystallography
promises to illuminate a broad range of biomolecules, from light
sensitive photoreceptors to other vital proteins.